reactions in GCs. The EFNB1-EPHB6 ligand-receptor pair regulate GC TFH recruitment and retention in a contact-dependent, repulsive manner.
This is unusual because contact-dependent guidance regulates embryonic tissue patterning that is
mainly driven by cell migration of the mesenchymal type, which requires cell-cell or cell-substrate
adhesion and often involves binary choices of
direction (24). Lymphocytes in the secondary
lymphoid organs typically migrate in an amoeboid
manner; are faster by orders of magnitude, with
a very relaxed requirement for substrate adhesion; and typically have a freedom of movement
in full or close-to-full three-dimensional space, a
situation that is well suited for guidance by diffusible directional cues (44, 45). By contrast, GCs are
unique tissue microdomains that are assembled
by antigen-specific B cells and present well-defined
borders. At the level of individual cells, GC B cells
present antigen and induce inside-out signaling
to integrins on TFH cells, which promotes T-B
adhesion, including the more dynamic form of
entanglement (27). EFNB1 and EPHB6 collaborate to inhibit such interactions and accelerate
de-adhesion of TFH cells from GC B cells. Although
a T cell that de-adheres from an interacting B cell
could move away in any spatial direction, de-adhesion at this individual level eventually manifests as territorial repulsion when B cells carrying
the repulsive EFNB1 form the GC aggregate and
claim the GC territory, essentially reducing the
spatial freedom of migrating TFH cells to binary
choices between entering or staying and leaving
at the tissue domain boundary.

Because antigen specificity is required for TFHresidence in GCs, negative regulation of GC re-tention of TFH cells may serve to select thosehelper T cells that carry more sensitive TCRs forprolonged GC residence (46, 47), which could pre-sumably facilitate affinity discrimination (48). Itmay also serve to protect TFH cells from beingexhausted by constant TCR stimulation insideGCs. Consistent with this notion, the EFNB1-EPHB6 axis appears to inhibit the retention morestrongly than recruitment of TFH cells (Fig. 2, Fand G, and fig. S4). We speculate that the level ofEPHB6 expression or the strength of its down-stream signaling might be kinetically regulated,such that individual TFH cells newly arriving atthe GC are minimally repulsed but, as the residencetime increases, become increasingly subjected tostronger repulsion that eventually pushes themout of the GC. It is not yet clear how EPHB6signals to effectuate TFH repulsion. EPHB6 lackskinase activities owing to conserved mutations ofits kinase domain (49, 50), but it could be trans-phosphorylated by other EPH receptors or theSrc-family Fyn kinase (51, 52). Evidence also sug-gests that EPHB6 could associate with the E3ubiqutin ligase c-Cbl (51, 53). These mechanisticuncertainties notwithstanding, fine-tuning of TFHretention time through territorial repulsion maybe a general organizational principle for GC TFHdynamics. We speculate that additional negativeregulators expressed by TFH cells might partici-pate in orchestrating this process.IL-21 is one of the most important cytokinesthat TFH cells produce to sustain GC B cell pro-liferation and promote continued output of plas-ma cells (12, 40–42). Our study identifies EFNB1as a niche factor that, by forward signaling throughEPHB4 and, to a lesser extent, EPHB6, promotesIL-21 production in TFH cells. Because all GC B cellsexpress a high level of EFNB1, GC TFH cells areessentially immersed in an environment that con-stantly drives their IL-21 production. However,it remains to be determined whether IL-21 issecreted by TFH cells as soon as it is produced or,perhaps more likely, the timing and directness ofthe cytokine secretion are additionally regulatedduring T-B contacts (54). The local EFNB1-mediatedregulation also suggests that the ability to produceIL-21 by T cells is not necessarily hard-wired byepigenetic programming but is ready to be modu-lated by the environment according to local tis-sue needs, at least in the case of GCs (2, 12, 40).Reduced IL-21 production from GC TFH cells isthe most likely reason for defective plasma cellformation in the absence of B cell–derived EFNB1,although our results do not rule out that EFNB1reverse signaling may still play a minor role orthat it can regulate other aspects of B cell biologyyet to be discovered.When dysregulated, GC reactions can lead to Bcell lymphomas and autoantibody-mediated dis-eases (55, 56). Thus, although it appears counter-intuitive that the same EFNB1 molecule wouldmediate both repulsive inhibition of GC TFHretention and positive promotion of TFH helpercytokine production, the self-contradicting natureof the EFNB1 regulatory axis probably contributeto ensuring that the potentially dangerous GC re-action is productive yet self-limiting.

Movie 5. GC recruitment and retention of
TFH cells transduced with EPHB6- targeting
shRNA. Seven days after HEL-OVA immunization of mice that were given dsRed-expressing
(red) OT-II Tcells that were transduced with an
EPHB6-knockdown vector and CFP-expressing
(blue) wild-type MD4 B cells, the draining
inguinal lymph node was imaged by two-photon
intravital microscopy. The image sequence was
acquired at one frame per 30 s. See quantitative
analyses in fig. S4. Scale bar, 100 mm.

Movie 6. GC recruitment and retention
of TFH cells transduced with control shRNA.

Seven days after HEL-OVA immunization
of mice that were given dsRed-expressing (red)

OT-II Tcells that were transduced with a control
shRNA vector and CFP-expressing (blue) wild-type MD4 B cells, the draining inguinal lymph
node was imaged by two-photon intravital
microscopy. The image sequence was acquired
at one frame per 30 s. See quantitative analyses
in fig. S4. Scale bar, 100 mm.